Click-modified mrna

ABSTRACT

The present invention relates to alkyne- and/or azide-modified mRNA, processes for producing such modified mRNA, cells which are transfected to include the modified mRNA, pharmaceutical compositions containing the modified mRNA or cells including the modified mRNA, and to uses of such mRNA, cells or pharmaceutical compositions in mRNA based therapeutic and/or prophylactic applications.

The present invention relates to alkyne- and/or azide-modified mRNA,processes for producing such modified mRNA, cells which are transfectedto include the modified mRNA, pharmaceutical compositions containing themodified mRNA or cells including the modified mRNA, and to uses of suchmRNA, cells or pharmaceutical compositions in mRNA based therapeuticand/or prophylactic applications. Finally, the invention relates to amethod of stabilizing RNA by introducing alkyne- and/or azide-modifiednucleotides and/or further to methods for determining delivery ofmodified mRNA into target cells and/or expression of a protein productencoded by the modified mRNA.

BACKGROUND OF THE INVENTION

Messenger RNA (mRNA) is the template molecule that is transcribed fromcellular DNA and is translated into an amino acid sequence, i.e. aprotein, at ribosomes in the cells of an organism. In order to controlthe expression level of the encoded proteins, mRNAs possess untranslatedregions (UTRs) flanking the actual open reading frame (ORF) whichcontains the genetic information encoding the amino acid sequence. SuchUTRs, termed the 5′-UTR and the 3′-UTR, respectively, are sections ofthe mRNA located before the start codon and after the stop codon.Further, mRNA contains a poly(A) tail region which is a long sequence ofadenine nucleotides which promotes export of mRNA from the nucleus,translation and to some extent protects the mRNA from degradation.

Due to its chemical and biochemical properties, mRNA usually is degradedwithin a few minutes inside of cells, thus expression of a specificprotein usually is a transient process. Moreover, the polyanionic mRNAmolecule is not well suited to cross a cell membrane which rendersexternal delivery of mRNA extremely difficult.

Despite these challenges associated with mRNA, scientific andtechnological advances of the recent years have made mRNA a promisingcandidate for a novel class of drugs. Sahin U. et al., Nat. Publ. Gr.13, 759-780 (2014) provide an overview on mRNA-based therapeutics anddrug development. mRNA is for example used to trigger in vivo productionof proteins like antibodies and enzymes, or to stimulate an immuneresponse, e.g. by expressing specific epitopes or via innate immuneresponse towards structural mRNA parts. For example, RIG-1 binds5′-triphosphate ends of RNA and triggers a signal cascade which resultsin activation of transcription factors and release of cytokines as partsof an antiviral response. Application of mRNA for stimulation of animmune response can be used in novel approaches to treat cancer, AIDSand to generate vaccines against almost any disease (cf. Pardi, N. etal., Nat. Publ. Gr. 543, 248-251 (2017) and Schlake T. et al., RNA Biol.9, 1319-30 (2012)). Key to these exciting developments is the robust invitro production of stabilized mRNA with improved translation efficiencyand its delivery into cells using special transfection formulations.

mRNA stability and translation efficiency depend on several factors.Especially the untranslated regions at either ends of the mRNA play acrucial role. In eukaryotic protein expression, a cap structure at the5′-end and the poly(A) tail at the 3′-end both increase mRNA stabilityand enhance protein expression. In addition, the 5′-UTR contains aribosome binding site necessary for translation and the 3′-UTR containsRNA sequences that adopt secondary structures which improve stabilityand influence translation. Moreover, modified natural, e.g.N1-methylpseudouridine, and artificial nucleotides can be incorporatedto improve mRNA stability and enhance translation of the mRNA (SvitkinY. V. et al., Nucleic Acids Research, Vol. 45, No. 10, 6023-6036(2017)).

Delivery of mRNAs into cells can be achieved by providing mixturescontaining lipids for fusion with the cellular membrane and cations toneutralize the negative charge of the oligonucleotide backbone. Specialformulations have been created to optimize mRNA delivery and to confersufficient in vivo stability for clinical trials. Most of the mRNAformulations which are applied intravenously are taken up by and areexpressed inside liver cells. This is due to the fact that the liverplays a major role in fatty acid metabolism and a high lipid content ofthe mRNA formulations therefore displays an organ-specific targetingeffect. In most cases the liver is, however, not the desired target andtherefore efforts are being made to modify lipid formulations to targetorgans, which are involved in an immune response, like e.g. the spleen(Kranz L. M. et al., Nature 534, 396-401 (2016)). Alternatively, cellsof the immune system (e.g. lymphocytes) can be isolated from a patients'blood and mRNA application is performed ex vivo to allow targeting. Mostrecently, tissue-specific targeting of mRNA using antibody fragmentmodified lipid formulations has been disclosed (Moffett H. F. et al.,Nat. Commun. 8, 389 (2017)).

Despite the recent advances and developments regarding the therapeuticapplicability of mRNA either directly or indirectly, i.e. via ex vivotransfection of cells and returning such transfected cells to a patient,further improving the stability of mRNAs and developing new options inthe context of their use as therapeutics or drugs are objects of ongoingresearch. Moreover, it is desirable to provide methods that allow for astreamlined and efficient production of therapeutic mRNA. Further, thereis still a need for advanced targeted delivery of mRNA for proteinsubstitution and gene replacement therapies, especially in the contextof the treatment of inherited diseases. It is also highly desirable toenable the monitoring of delivery and protein expression. Finally,exploring further options for exploiting the immune stimulatory effectof mRNAs in e.g. cancer therapy is another object of ongoing research.

SUMMARY OF THE INVENTION

The present invention is directed to providing solutions to theabove-mentioned objects and relates inter alia to a new kind of mRNAmodification. Such modification not only allows to stabilize mRNAs ofinterest for ex vivo application and for subsequent administration to ahuman patient, animal or plant, but also to easily attach detectablelabels or functional groups which e.g. allows for targeted delivery ofthe modified mRNA to specific cells or tissues and to monitor suchdelivery.

In a first aspect, the present invention relates to modified mRNA whichcomprises a 5′-cap structure, a 5′-untranslated region (5′-UTR), an openreading frame region (ORF), a 3′-untranslated region (3′-UTR) and apoly(A) tail region, wherein the mRNA contains at least one of analkyne- or azide modification in the nucleotides within at least one ofthe ORF, the 5′-UTR, the 3′-UTR and the poly(A) tail region. Inespecially preferred embodiments of this first aspect of the presentinvention, the modified mRNA contains one or more of a detectable labeland/or a functional molecule introduced via a click reaction of themodified mRNA with a correspondingly modified alkyne- orazide-containing detectable label or functional molecule.

In a second aspect, the invention relates to a process for producing themodified mRNA according to the present invention, wherein such processcomprises in vitro transcription of mRNA from a DNA template in thepresence of an RNA polymerase and a nucleotide mixture containing thenucleotides required for RNA transcription, wherein at least a part ofthe nucleotides in the nucleotide mixture is modified to contain analkyne- or azide-modification at the nucleotide. In an alternativeembodiment of this second aspect, the modified mRNA is produced via afermentation process. In such process, eukaryotic or prokaryotic cellsare transformed to contain the genetic information (e.g. plasmid) forproducing the desired mRNA and alkyne- or azide-modified nucleosides,nucleotides or nucleotide prodrugs are included in the growth medium. Inanother alternative embodiment of this second aspect, the mRNA of theinvention is produced synthetically, via solid phase or phosphoramiditesynthesis.

A third aspect of the present invention relates to an enzymatic methodfor the preparation of a site-specifically modified mRNA of theinvention which contains an alkyne- or azide-modification in a definedregion of the mRNA, e.g. the poly(A) tail region only. Such processcomprises performing a poly(A) polymerase addition reaction on an mRNAin the presence of adenosine triphosphate (ATP), wherein the ATP is atleast partly alkyne- or azide-modified at the nucleotide.

In especially preferred embodiments of the second and third aspects ofthe invention, one or more of correspondingly alkyne- or azide-modifieddetectable labels and/or functional molecules are added under conditionsto perform a click reaction to produce a modified mRNA comprising suchdetectable label(s) or functional molecule(s).

A fourth aspect of the present invention relates to a cell preparation,especially a preparation of cells of the immune system, which containsthe modified mRNA of the present invention and is obtained by ex vivotransfection.

A fifth aspect of the present invention relates to pharmaceuticalcompositions which comprise as an active agent or as an immunologicadjuvant a modified mRNA of the present invention or a cell preparationwhich was obtained by ex vivo transfection to include such mRNA.

A still further and sixth aspect of the present invention is a modifiedmRNA according to the invention, of a cell preparation including suchmRNA or of a pharmaceutical composition of the invention for use inmRNA-based therapeutic and/or prophylactic applications in a human or ananimal.

The use of a modified mRNA of the present invention for transfectingplants or plant cells is a further, seventh aspect of the invention.

An eighth aspect of the invention relates to diagnostic compositions forin vitro or in vivo screening for the presence, delivery and/ordistribution of the inventive mRNA in cells, tissues or organs, suchcompositions comprising a modified mRNA of the present inventioncontaining or afterwards being modified with a detectable label,preferable a fluorophore or a radionuclide.

A ninth aspect of the invention relates to a kit of parts for preparingand/or delivering a modified mRNA of the present invention. Inespecially preferred embodiments, such kit also contains one or more ofcorrespondingly alkyne- or azide-modified detectable labels orfunctional molecules to obtain modified mRNAs containing such detectablelabels and/or functional molecules upon performing the click reactionbetween modified mRNA and modified label/functional molecule.

A tenth aspect of the present invention relates to a method forstabilizing RNA, especially mRNA, wherein an alkyne- and/orazide-modification is introduced by including at least one of the fourstandard types of nucleotides (ATP, CTP, GTP and UTP) and/or anotheralkyne- or azide-modified compatible nucleotide or pseudonucleotide(i.e. a nucleotide with false or unusual structure as compared to thestandard types of nucleotides) in partly or completely alkyne- and/orazide-modified form during RNA synthesis and/or in a poly(A) polymeraseaddition reaction. A further stabilization can be obtained by couplingof the corresponding azide- and/or alkyne-modified molecules or groupsto the modified RNA via a click reaction.

An eleventh aspect of the present invention is a method forqualitatively and quantitatively determining at least one of thedelivery to and expression of an mRNA of the present invention in atransfected cell via fluorescence-activated cell scanning (FACS).

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

The present invention employs so-called “click chemistry” or elementsthereof and applies this technique to modify mRNA molecules to impartimproved stability and/or to provide for use of such modified mRNAmolecules in the context of inter alia mRNA based therapy and mRNAvaccine technologies.

Click chemistry is a concept which was defined in 2001/2002 by thegroups of Sharpless and Meldal (Sharpless, K. B. et al., Angew. Chem.2002, 114, 2708; Angew. Chem. Int. Ed. 2002, 41, 2596; Meldal, M. etal., J. Org. Chem. 2002, 67, 3057). Since then, especially the coppercatalyzed reaction of azides with alkynes to give 1,2,3-triazoles, avariation of the 1,3-dipolar Huisgen cycloaddition (R. Huisgen,1,3-Dipolar Cycloaddition Chemistry (Ed.: A. Padwa), Wiley, New York,1984), has become a very widely used method to perform a click reaction.As a result of its mild conditions and high efficiency, this reactionhas found a myriad of applications in biology and material sciences,such as e. g. DNA labeling for various purposes (Gramlich, P. M. A. etal., Angew. Chem. Int. Ed. 2008, 47, 8350).

In addition to the copper-catalyzed click-reaction, also copper-free,bio-orthogonal methods have been developed and all such methods cangenerally also be employed in the context of the present invention.E.g., strain-promoted azide-alkyne cycloaddition (SPAAC) (I. S. Marks etal., Bioconjug Chem. 2011 22(7): 1259-1263) can be used either alone orin combination with copper-catalyzed click chemistry (CuAAC) in thecontext of the present invention. Especially in cases in which it isdesirable to perform a labelling reaction in vivo in cell culture or ina living organism, performing such reaction using SPAAC is preferablesince the method does not require the use of toxic substances orexternal catalysts.

Click chemistry facilitates attaching reporter molecules or labels tobiomolecules of interest and is a very powerful tool for identifying,locating, and characterizing such biomolecules. The method for exampleenables inclusion and attachment of fluorescent probes for spectrometricquantification, or of anchor molecules to allow for separation andpurification of the target biomolecules. Up to date, many applicationshave been developed in which click chemistry is used as an underlyingprinciple. Next-generation sequencing is one of such applications whichbenefits from this technique where formation of so-called “backbonemimics”, i.e. non-natural alternatives for the phosphodiester bond,which can be generated by copper-catalyzed azide alkyne cycloaddition(CuACC), is used to ligate e.g. DNA fragments and adapter sequences.Despite the presence of a triazole ring instead of a phosphodiesterbond, such backbone mimics are acceptable substrates for polymerasedriven DNA or RNA preparation methods like PCR or reverse transcription.Detection of cell proliferation is a further field of application forclick-chemistry. The methods that are normally applied include addingeither BrdU or radioactive nucleoside analogs to cells duringreplication and detecting their incorporation into DNA. Methodsinvolving radioactivity, however, are rather slow and not suitable forrapid high-throughput studies and are also inconvenient because of theradioactivity involved. Detecting BrdU requires an anti-BrdU antibodyand applying denaturing conditions resulting in degradation of thestructure of the specimen. The development of EdU-click assays hasovercome such limitations by including 5-ethynyl-2′-deoxyuridine, athymidine analog, in the DNA replication reaction. The detection viaclick chemistry instead of an antibody is selective, straight forward,bioorthogonal and does not require DNA denaturation for the detection ofthe incorporated nucleoside.

Within the context of the present invention it was discovered that it ispossible to introduce alkyne- and/or azide-modified nucleotides duringin vitro transcription of mRNA or during a fermentation process forproducing mRNA to result in a correspondingly modified mRNA. The alkyne-or azide-modification can be included in only some or all elementscontained in the mRNA, and needs to be included in at least one of theUTRs, ORF and poly(A) tail. The 5′cap structure preferably does notcontain such alkyne- or azide modifications, as changes to the capstructure can interfere with efficient binding of initiation factorslike eIF4E, eIF4F and eIF4G and thus drastically decrease translationefficiency. The presence of such modification on the one hand stabilizesthe mRNA and on the other hand provides specific anchor sites forpost-enzymatic labeling or for attachment of tissue- or cell-specificligands or targeting molecules via click chemistry. Thus, the presentinvention not only enables detection of the presence and the location ofmRNA after transfection or application, but also provides new optionsfor targeted delivery of mRNAs to specific organs or cell types in thecontext of therapeutic applications. A correspondingly modified mRNA isa first subject matter of the present invention.

Depending on which type of nucleotide or nucleotides are included inalkyne- or azide-modified form during in vitro transcription or duringmRNA production in prokaryotes or eukaryotes via fermentation, theresulting modified mRNA can contain modifications not only in the 5′-and 3′-UTRs and the ORF, but also in the poly(A) tail region. Asapparent to the skilled person, including e.g. one or more of modifiedCTP, GTP and UTP leads to a modification within the UTRs and the ORF,while additionally including modified ATP results in a modification alsoof the poly(A) tail region. Including only alkyne- and/or azide-modifiedATP during the transcription leads to modifications in the UTRs, the ORFand the poly(A) tail region.

No severe negative effects caused by the presence of alkyne- orazide-modified nucleotides within the mRNA of the invention have beenobserved. Depending on the amount of modified nucleotides included inthe reaction, the in vitro and in vivo transcription efficiency can beas effective as in cases where only non-modified nucleotides are presentin the reaction mixture, or slightly decreased. Furthermore, themodification of the mRNA does not seem to impair translation of mRNAduring protein production at the ribosomes. Depending on thecircumstances, the amounts of modified nucleotides to be included in thein vitro transcription reaction or the fermentation process can beadjusted to either provide maximum mRNA yield or maximum modification.For instance, when a dye is to be attached to the mRNA as a detectablelabel via a click reaction, it might be desirable to include anadequately high amount thereof to ensure and facilitate detection,whereas in order to target specific cell receptors, it might besufficient to include only one or a few respective ligand molecules toachieve the desired effect.

As will be explained later in more detail, including alkyne- orazide-modified nucleotides has a stabilizing effect on mRNA. It is to beexpected that the stabilizing effect of the inventive modification ismost pronounced if such modification is distributed over the completemRNA molecule. In such case, subsequent attachment of detectable labelsand/or functional molecules via click chemistry will occur alsouniformly over the whole mRNA molecule and can even provide for anenhanced stabilizing effect.

However, in some cases it may be important to restrict the inclusion oflabels or functional molecules to a part of the mRNA molecule which isnot involved in subsequent translation of mRNA during proteinexpression. For such purpose, it can be desirable to include modifiednucleotides in the poly(A) tail region only whereby it can be ensuredthat ribosomal activity is not impaired by the presence of especiallylonger or bulkier labels or functional molecules like ligands ortargeting molecules.

The present invention therefore also provides a modified mRNA containingalkyne- or azide-modification in the poly(A) tail region only. Insteadof including alkyne- or azide modified nucleotides during in vitrotranscription of a DNA template or a fermentation process, amodification in only the poly(A) tail region can be achieved for anydesired mRNA by performing an addition reaction in the presence ofpoly(A) polymerase and alkyne- or azide-modified ATP.

By controlling the amount and type of alkyne- or azide-modification inthe modified mRNA of the invention it is possible to conveniently andeasily adapt the resulting mRNA to impart stabilization and options forpost-enzymatic attachment of molecules of interest as required andviable with regard to any intended application.

Within the context of the present invention the azide- oralkyne-modification can be included at the nucleobase or at the2′-position of the ribose unit of the respective nucleotide. In veryspecial cases, inclusion of a nucleotide containing the modification atthe 3′-position of the ribose is also possible. In such case, theenzymatic poly(A) addition reaction is terminated upon inclusion of onemodified nucleotide. In one preferred embodiment of this aspect of thepresent invention, the modified mRNA contains an alkyne- and/or anazide-modification at the nucleobase or the 2′-ribose position in atleast one of nucleotides within at least one of the UTRs, the ORF andoptionally also the poly(A tail region, and additionally achain-terminating alkyne- or azide-modification at the 3′-position ofthe ribose in the poly(A) tail. In a different preferred embodiment, themRNA of the present invention does not contain a chain-terminatingalkyne- or azide-modification at the 3′-ribose position in the poly(A)tail region.

The modified nucleotide included in the mRNA of the present inventioncan be derived from a natural nucleotide and especially one of thestandard nucleotides with adenine, cytosine, guanine or uracil bases, orit can be a modification of another naturally occurring nucleotide (e.g.pseudouridine derivative) or even a non-naturally occurring molecule(e.g. F. Eggert, S. Kath-Schorr, Chem. Commun., 2016, 52, 7284-7287)which does not negatively affect transcription and/or translation andthe function of the resulting modified mRNA. Preferably the modifiednucleotide is derived from a natural nucleotide or a naturally occurringnucleotide within mRNA.

Suitable alkyne- and azide-groups for click reactions are known andavailable to the skilled person and all such groups can be used toprepare modified nucleotides and modified mRNAs in the context of thepresent invention. The alkyne-modified nucleotide preferably is anethynyl-modified nucleotide, more preferably 5-ethynyluridine phosphateor 7-ethynyl-7-deazaadenine phosphate. While it is in principle alsopossible to employ higher alkyne-modified nucleotides, especiallypropynyl or butynyl modified nucleotides and even C—C triplebond-containing ring systems, possible negative effects on e.g. thetranscription or poly(A) polymerase reaction efficiency as well as on afurther translation of the mRNA into a protein will have to beconsidered when selecting suitable alkyne molecules. Azido-modificationsfor nucleotides which are useful in the present invention can, e.g.,also include azidoalkyl groups in which the alkyl part preferably is alower alkyl group, especially a methyl, ethyl or propyl group. As anazide-modified nucleotide, preferably 5-(3-azidopropyl)-uridinephosphate or 8-azidoadenine phosphate are considered for inclusion inthe inventive mRNA. An example for an azide-modified nucleotide causingtermination of the poly(A) addition reaction is 3′-azido-2′,3′-dideoxyadenine phosphate.

In principle, all nucleotides of the at least one type of modifiednucleotide can be alkyne- or azide-modified, or alternatively, only apart of such nucleotides is present in modified form. In a preferredembodiment of the present invention and depending on the desiredmodification and modification rate, ratios of modified versusnon-modified forms of the various nucleotides can range from 1:100 to10:1, preferably 1:10 to 10:1, further preferably 1:4 to 4:1, and alsopreferably 1:2 to 2:1. Preferably, a 1:1, 1:4 or 1:10 combination ofmodified to non-modified nucleotide is included in the mRNA of theinvention.

As mentioned above, the presence of alkyne- or azide-modifiednucleotides or nucleobases in the modified mRNAs of the presentinvention confers a stabilizing effect. On the one hand, the attack ofendoribonucleases is restricted to some extent by an internalmodification. Extension of the poly(A) tail region during poly(A)polymerase based addition of modified ATPs at the 3′-end leads to afurther stabilizing effect. Attack on and degradation of mRNA moleculesby exoribonucleases occurs at the two ends of RNA. The mRNA according tothe invention contains a cap at the 5′-end which provides protectionfrom degradation at that side. Including additional modified adenosinenucleotides at the 3′-end imparts further protection as the attack ofexoribonucleases in 3′→5′ direction is impeded and degradation reachingthe core mRNA, especially the ORF, is delayed.

In a preferred embodiment of the present invention, detectable labelsand/or functional molecules can be introduced into the modified mRNA viaclick reaction with a correspondingly modified alkyne- orazide-containing label or functional molecule. As for the nucleotidemodification, also for the modification of detectable labels orfunctional groups, suitable alkyne- and azide groups are known to theskilled persons and the preferred examples for such groups areapplicable as described above. The reaction of an alkyne-modifiednucleotide within the modified mRNA and an azide-containing label orfunctional molecule or the reaction of an azide-modified nucleotidewithin the modified mRNA of the invention and an alkyne-containing labelor functional molecule is performed under conditions to conduct theclick reaction and leads to formation of the 5-membered heterocyclic1,2,3-triazole moiety which forms the link between mRNA and label orfunctional molecule. According to the present invention, the termalkyne-containing label or functional molecule also encompasses C—Ctriple bond-containing ring systems like cyclooctynes which have beenconsidered especially in the context of SPAAC reactions and in vivolabelling via bio-orthogonal ligation reactions.

The type and size of labels and functional molecules are notparticularly restricted and, again, are determined by the intended use.Preferred examples for the detectable label include color imparting or afluorescence imparting labels, e.g. fluorescein derivatives like FITC,Alexa Fluor dyes or DyLight Fluor dyes, cyanine dyes like Cy5 and Cy3 orrhodamine dyes like Texas Red and 5-TAMRA, or any other fluorescent dye.Even non-colored small molecules can be used (e.g. biotin), when theyare substrates for an enzyme or a binding protein-enzyme conjugate (e.g.antibody enzyme conjugates) and can produce a colored or luminescentproduct through an enzymatic reaction cascade and a further substrate.Also, radionuclides can be included as detectable labels, e.g.preferably positron emitting radionuclides which can be detected usingpositron emission tomography scan. For radionuclides with shorthalf-lifes, e.g. ¹⁸F, the ability of quick and robust labeling of mRNAusing a post mRNA production click labeling could be the only feasiblemethod to obtain material for mRNA biodistribution studies using PET.Depending on the intended use, also heavy isotopes like C¹³ or P³³ canbe considered as a detectable label for the present invention.

Functional molecules to be included in the modified mRNA via a clickreaction are not restricted and are preferably cell- or tissue-specificligands that mediate targeted uptake of the mRNA into specific tissuesor cells including cancer cells or at least allow to attach or anchorthe mRNA onto the cell-surface. Such cell- or tissue-specific targetingcan be achieved for example by using specific antibodies or antibodyfragments, peptides, sugar moieties, small molecules (e.g. folic acid)or fatty acid moieties as the cell- or tissue-specific ligands.Respective substances have been described for a large number oftargeting applications and are available to the skilled person. Somepreferred and exemplary targeting molecules are antibodies or antibodyfragments or receptor ligands which target cell specific receptors likee.g. the epidermal growth factor receptor, folate which targets thefolate receptor, apolipoproteins which target endogenous low-densitylipoprotein receptors or arachidonic acid which targets the endogenouscannabinoid receptors. Also, the amino acid sequence RGD or similarsequences have been found to mediate cell adhesion and can also beconsidered as preferred ligands within the context of the presentinvention.

The presence of functional molecules attached to the mRNA can furtherincrease mRNA stability against nuclease degradation and it has beenshown that partial as well as full replacement of at least one of thenatural nucleotides within the mRNA for an alkyne- or azide-modifiedanalogue and even attachment of functional molecules thereto does nothamper translation of the mRNA molecule.

In addition to including either alkyne-modified or azide-modifiednucleotides, it is also possible that an inventive modified mRNAcontains at least one nucleotide in partially or completelyalkyne-modified form and at least one other nucleotide in partially orcompletely azide-modified form. A further option is an mRNA including atleast one type of nucleotide in partially or completely alkyne- as wellas in partially or completely azide-modified form. Such mRNA containstwo different anchor modifications to which different labels orfunctional molecules can be attached in a downstream post-enzymaticclick reaction. For example, but without limiting to such specificembodiment, an alkyne-modified cell-specific targeting group as well asan azide-modified detectable label can then be attached resulting inanother preferred embodiment of a modified mRNA according to the presentinvention.

It is also possible and preferred within the context of the invention toprovide a modified mRNA containing at least an azide-modified nucleotideand an alkyne-modified nucleotide, wherein e.g. at the azide-modifiednucleotide a detectable label or a functional molecule has been attachedvia a biorthogonal reaction, e.g. SPAAC in vitro, whereas thealkyne-modified nucleotide is available for a downstream in vitrolabelling reaction via a CuAAC reaction. If CuAAC reaction conditionsare applied to the double labeled mRNA (containing alkyne and azidefunctions), it is possible to circularize the mRNA, which is, e.g., avaluable alternative to using self-splicing introns (DOI:10.1038/s41467-018-05096-6).

It is for example also conceivable for the inventive modified mRNA tocontain one kind of modification in the UTRs and the ORF and anothermodification solely in the poly(A) tail. Such modification can beeffected by performing first a transcription reaction to introduce oneor more first types of modified nucleotide and then following up with apoly(A) polymerase reaction using a second type ofmodification-containing ATP.

It will be apparent to the skilled person that numerous modificationsand combinations of modifications are possible in the context of thepresent invention. Further, it is also possible to include differentlabels or functional groups based on the presence of the alkyne- and/orazide-modifications on the mRNA molecule, but rather also by consecutiveaddition under click reaction conditions of different appropriatelymodified labels or functional molecules. Consequently, the presentinvention provides a vast number of options and a convenient modularityin order to adapt the modified mRNA to the intended use in an optimalmanner.

Apart from including alkyne- and/or azide-modified nucleotides, thepresent invention generally also allows for other modifications in thenucleotides as far as such other modifications do not adversely affectmRNA production or the intended use of the resulting mRNA to an extentwhich is not acceptable when contemplating the intended use (i.e. themodification is compatible with the modified mRNA within the context ofthe invention). As an example of such other modified nucleotide ornucleotide derivative that can be included in the mRNA,pseudouridine-5′-triphosphate (pseudo-UTP) can be considered.Pseudouridine (or 5′-ribosyluracil) was the first modifiedribonucleoside that was discovered. It is the most abundant naturalmodified RNA base and is often designated as the “fifth nucleoside” inRNA. It can be found in structural RNAs, such as transfer, ribosomal andsmall nuclear RNA. Pseudouridine has been shown to enhance base stackingand translation. Further, pseudouridine-5′-triphosphate is able toimpart advantageous mRNA characteristics such as increased nucleasestability and altered interaction of innate immune receptors with invitro transcribed RNA. Incorporation of pseudo-UTP and also furthermodified nucleotides, like N1-methylpseudouridine and5-methylcytidine-5′-triphosphate into mRNA, have been shown to decreaseinnate immune activation in culture and in vivo while simultaneouslyenhancing translation (B. Li et al., Bioconjugate Chemistry, 2016, 27,849-853 and Y. Svitkin et al., Nucleic Acid Research, 2017, 45,6023-6036). Inclusion of these and other suitable and compatiblenucleotides, nucleotide analogues or non-naturally occurring moleculesas described earlier in this specification, in alkyne- or azide-modifiedor in non-modified form is therefore a further option and preferredembodiment of the present invention.

As apparent from the above description of the modified mRNA of thisinvention, a multitude of different options exist to prepare or adapt anmRNA molecule to be beneficially applicable for various purposes. Theinvention is not restricted to a particular type of mRNA, which canrather be chosen in accordance with any intended use thereof, especiallyin the applications described in general or in more detail above in thebackground section as well as in the following. The mere introduction ofthe alkyne- or azide-modification conveys enhanced stability to an mRNAmolecule which can be administered to deliver genetic information forapplications like protein replacement therapy or to deliver mRNA forimmunostimulatory purposes or as an mRNA-vaccine. Further modifying themRNA via downstream click-coupling of respectively modified labels orfunctional molecules provides further possibilities especially forscreening delivery of the modified mRNA and/or to target delivery of themRNA to specific cells or tissues e.g. in a gene replacement therapy orto improve pharmacokinetics (e.g. slower renal clearance by adding PEGlabels).

Within the context of the present invention, the modified mRNA of thepresent invention can encode a functional protein of interest.Furthermore, the modified mRNA of the invention can encode a recombinantprotein like a chimeric protein or any further combination of proteins,peptides or peptides and proteins which can be advantageously used for adesired purpose. Especially an mRNA encoding a recombinant fusionprotein, e.g. an mRNA comprising a sequence encoding a first protein orpeptide ligated in frame with a sequence encoding a second protein orpeptide are considered within the context of the present invention. Thesecond protein or peptide can, e.g., target a specific localizationwithin a cell or a tissue. Especially when considering the monitoring ofthe delivery and the localization of the modified mRNA of the inventionor of a protein encoded by the mRNA within a target cell, a fusionprotein of the protein of interest with a reporter protein like thegreen fluorescent protein (GFP), the enhanced green fluorescent protein(eGFP) or with a protein or peptide tag, e.g. the snap tag, isconsidered as a further preferred embodiment of the invention. To thispurpose, the modified mRNA of the present invention can be engineered toexpress the fusion protein as a single protein preferably including twoor more different functions as exemplarily outlined above. By means ofincluding linkers, spacers or cleavage sites for proteases, productionof two or more separate proteins is equally conceivable.

In the preferred embodiment of expression of a fusion protein of aprotein of interest and GFP or eGFP, localization of the fusion proteincan easily be detected under a fluorescent microscope using appropriatefilters. Further, detection and quantification of transfected cells andproduction of the protein is also possible via other methods, preferablyvia flow cytometry, especially fluorescence-activated cell sorting(FACS). Using the above-mentioned methods allows for a qualitative andquantitative screening for cells which include a fluorescent moleculewhich especially is either a label introduced via a click reaction or apeptide or protein (co-)encoded by the mRNA itself.

Within the context of the present invention, the modified mRNA of thepresent invention can be used together with substances which arerequired or preferably present for a certain application. For example,for ex vivo cell transfection but also for in vivo administration,substances which facilitate mRNA uptake by cells are preferably combinedwith the mRNA. Lipid formulations as well as nanocarriers (e.g. asdescribed by Moffett et al., mentioned supra) can preferably be includedin respective compositions and formulations within the context of thepresent invention. Accordingly, a mixture of substances containing themodified mRNA and at least one other substance as mentioned above, or akit of parts in which the modified mRNA and at least one other suitablesubstance are provided in different containers for subsequent combineduse are further subjects of the present invention.

When combined with one or more other active substances, especially oneor more substances which trigger an adaptive immune response, themodified mRNA of the present invention can also act as an adjuvant toenhance an innate immune response and, thus, an overall immunogeniceffect. The effectiveness of substances like e.g. protein- orpeptide-based tumor vaccines benefits tremendously from beingadministered together with RNA adjuvants (e.g. Ziegler et al., J.Immunol. Jan. 11, 2107, 1601129;DOI:https://doi.org/10.4049/jimmunol.1601129, or by Heidenreich et al.,Int. J. Cancer. 2015 Jul. 15; 137(2):372-84, D01:10.1002/ijc.29402). Thefurther advantages which are inherent to the inventive modified mRNA asdescribed above in detail, ensure that the adjuvant properties of amodified mRNA of the present invention are comparable or even morepronounced than for non-modified RNA, while the stability of themolecule is improved and further options like targeted delivery orinclusion of labels via click reaction open up further perspectives.

For the adjuvant application mentioned above, the modified mRNA of thepresent invention can be combined or complexed with other substanceswhich are known to the skilled person as optional or mandatory in thiscontext, preferably cationic or polycationic compounds (see e.g.WO2010/037408). Complex formation or combination with such othersubstance confers improved immunostimulatory properties and especiallythe complex formation with a cationic element provides for aparticularly strong adjuvant effect and thus is considered a preferredembodiment of the invention.

When intended as an adjuvant, the mRNA of the present invention is notnecessarily required to encode a functional protein or peptide, ratheralso such non-coding RNAs which contain an alkyne- or azide-modificationand optionally further a functional molecule or a detectable labelintroduced via the click reaction, are included in the invention forthis purpose.

WO2010/037408 describes an immunostimulatory composition comprising anadjuvant component comprising at least one (m)RNA preferably complexedwith a cationic or polycationic compound, and at least one free (i.e.non-complexed) mRNA which encodes at least one therapeutically activeprotein, antigen, allergen and/or antibody.

In this context, while a modified mRNA of the present invention can beincluded as only the adjuvant component, also a combination of amodified (m)RNA of the present invention acting as adjuvant and afurther modified mRNA of the invention to be translated into a protein,antigen, allergen and/or antibody can be combined. Also, for such uses,it can not only be taken advantage of the possibility for specifictargeting and delivery to cells provided by the present invention butalso of the stabilization conferred to the (m)RNAs by the modificationas disclosed earlier herein.

Another subject of the present invention is a process for producing themodified mRNA of the present invention. According to a first process,mRNA is transcribed in vitro from a DNA template in the presence of anRNA polymerase, usually T3, T7 or SP6 RNA polymerase, and a nucleotidemixture containing at least the four standard types of nucleotides (ATP,CTP, GTP, UTP) required for mRNA transcription and optionally naturallyoccurring modified nucleotides, like e.g. N1-methylpseudouridinetriphosphate, or even suitable artificial nucleotides. In addition, toimprove the translation efficiency it is important to generate a 5′-capstructure, e.g. 7-methylguanylate for eukaryotes. At least a part of atleast one of the standard nucleotides, naturally occurring modifiednucleotide analogue or suitable artificial nucleotide analogues ismodified to contain an alkyne- or azide-modification at the nucleotide.

Depending on which type of nucleotide is used for the process, themodification will be effected in the UTRs and the ORF only (for modifiedCTP, GTP or UTP, or their analogues) or in all of the UTRs, the ORF andthe poly(A) tail (for modified ATP alone or in combination with one ormore of modified CTP, GTP or UTP, or their analogues).

The conditions and methods to perform in vitro mRNA transcription (IVT)as well as a poly(A) polymerase addition reaction are well known to theskilled person (e.g. Cao, G. J et al, N. Proc. Natl. Acad. Sci. USA.1992, 89, 10380-10384 and Krieg, P. A. et al., Nucl. Acids Res. 1984,12, 7057-7070)

Such conditions and methods are not particularly critical as long as asatisfactory yield of modified mRNA is obtained. In this context, alsothe kind of DNA template used within the first described process is notparticularly critical. Usually, DNA to be transcribed is included in asuitable plasmid, however it can also be used in linear form.Additionally, a DNA template usually contains a promoter sequence,especially a T3, T7 or SP6 promoter sequence.

During the process of producing the modified mRNA of the presentinvention, the obtained mRNA is preferably capped using well-knownmethods (Muthukrishnan, S., et al, Nature 1975, 255, 33-37). Requiredreactants for the capping are commercially available, for exampleA.R.C.A. (P1-(5′-(3′-O-methyl)-7-methyl-guanosyl) P3-(5′-guanosyl))triphosphate, a cap analog) (Peng, Z.-H. et al, Org. Lett. 2002, 4(2),161-164). Preferably, as an alkyne-modified nucleotide, ethynyl-modifiednucleotides, most preferably 5-ethynyl UTP or 7-ethynyl-7-deaza ATP, areincluded in the process. As an azide-modified nucleotide, preferably5-(3-azidopropyl) UTP, 3′-azido-2′,3′-dideoxy ATP (at the 3′-end only)or 8-azido ATP is used.

Within the context of the present invention, it is preferred to performthe transcription process using T7 RNA polymerase and to provide the DNAtemplate in a suitable vector for efficient template production usingmicroorganisms and subsequent in vitro transcription after linearizationof the vector.

As an alternative to in vitro transcription, also a fermentation processin prokaryotic or eukaryotic systems for producing the mRNA of theinvention is included in the context of the present invention. For thispurpose, a DNA template, which is usually included in a suitableexpression vector, preferably a plasmid containing the DNA of interestunder control of an RNA polymerase promoter, is introduced into hostcells or microorganisms and respective nucleosides or nucleotideprodrugs (to allow sufficient cellular uptake) as described above areincluded in the culture medium. Fermentative RNA production is known tothe skilled person, cf. e.g. Hungaro et al. (J Food Sci Technol. 2013October; 50(5): 958-964).

For illustration purposes, however not to restrict to such specificprocess, the production of alkyne-, azide- and click-modified mRNA viafermentation is described in more detail for a bacterial system: A DNAtemplate, encoding the mRNA of interest under control of an RNApolymerase promotor, is introduced into bacterial cells. Preferably thisis done via transfection of a plasmid. The design of the sequence isimportant and preferably contains all of several elements necessary forproduction of the desired mRNA: RNA polymerase promotor (e.g. T7 or SP6promoter); the open reading frame of interest (ORF); and preferably alsoa sequence encoding the poly(A) region (preferably 100-120 nt long).Moreover, the plasmid contains an origin of replication and a selectionmarker for controlled growth and amplification in cell culture. It ispreferable to have a gene regulatory element for the open reading frame,e.g. a lac operon, to selectively induce expression of the mRNA uponaddition of an external compound. Important is the poly(A) region,necessary for discrimination of the mRNA from all the other RNAs (e.g.bacterial mRNAs, tRNAs and rRNAs) during purification and to provide themRNA product with sufficient stability and translation efficiency. Apoly(A) tail region can, however, also be introduced or a comparativelyshort poly(A) tail can be extended and possibly also modified viapolymerase A addition reactions as described within the context of thisinvention after the fermentative production of the mRNA.

Alkyne- or azide modified nucleosides are added to the growing mediumand are taken up by the bacterial cells via transporters or passivemechanism (J. Ye, B. van den Berg, EMBO journal, 2004, 23, 3187-3195).Intracellularly these nucleosides are phosphorylated by kinases to thecorresponding triphosphates and can be incorporated into the mRNA. Sincethe monophosphorylation of the nucleosides is a slow process, it ispossible to feed monophosphate prodrugs of the nucleosides to increaseintracellular nucleotide concentrations (like for sofosbuvir).

In case of azide-modified mRNA, a click-reaction using biorthogonalchemistry, e.g. strain promoted azide-alkyne cycloadditions (SPAAC) canbe performed in cell culture.

Therefore, preferably cyclooctyne modified tags/labels or functionalmolecules are added to the medium.

The newly synthetized mRNA, which includes the modified nucleosideswithin the sequence, is then e.g. purified by the usage of poly(T)oligonucleotides attached to a specific resin and/or beads. e.g. of themRNA isolation kit form Sigma Aldrich (cat No: 000000011741985001).

It is well known that the mRNA of prokaryotic cells does not contain apoly(A) region or when it does it is not longer than 20 nt, which is notenough to be taken up by the poly(T) oligonucleotides attached to theresin and/or beads, thus allowing for an efficient separation of thedesired mRNA form prokaryotic mRNA. Thus, a fermentatively produced mRNAwithout a poly(A) tail regions or without a sufficiently long poly(a)tail region needs to be purified by other known methods via chloroformphenol extraction, precipitation and subsequent purification of thecrude cellular RNA by ion exchange chromatography.

The bacterial strain, e.g. E. coli BL21(DE), needs to have the RNApolymerase, e.g. the T7 RNA polymerase, integrated in the genomic DNA(e.g. DE3 strains). Production of the mRNA is then possible when aplasmid containing the T7 promotor is transformed and can introduce thealkyne- or azide modified nucleoside during in vivo transcription withinthe bacterial cell.

It is well known that the prokaryotic mRNA is lacking the 5″CAPstructure. This important element of the inventive modified mRNA can beintroduced after the purification of the mRNA or it can be introducedconcurrently by co-transforming the bacterial cell with another plasmidexpressing the eukaryotic capping enzyme.

As a further alternative, it is possible to produce the modified mRNA ofthe present invention via solid phase or phosphoramidite synthesis andinclude modified nucleotides as described above. Especially in caseswhere the (m)RNA is intended for use as an adjuvant and shortermolecules or non-coding sequences can be considered for such purpose,synthetic preparation can be convenient and effective. Respectivemethods are available to the skilled person and described e.g. inMarshall, W. S. et al. Curr. Opin. Chem. Biol. 2004, Vol. 8, No. 3,222-229.

A second process within the context of the present invention allows formodification of the poly(A) tail region only, by first providing an mRNAof interest by any suitable method and adding modified alkyne- orazide-modified ATP (or analog) in a poly(A) polymerase additionreaction. Such poly(A) polymerase addition reactions and suitableconditions are well-known to the skilled person and respective reactionkits are commercially available.

While the first and the second process described above can be usedseparately to provide modified mRNA of the present invention, it is alsopossible to use a combination of in vitro transcription or syntheticmRNA production and poly(A) polymerase addition reaction to includemodified alkyne and/or azide-modified nucleotides in the UTRs, the ORFand the poly(A) tail during the mRNA transcription step. By additionallyperforming the second process, i.e. a poly(A) polymerase additionreaction, a further extension of the poly(A) tail can be achieved,wherein ATP is at least partly included in an alkyne- or azide-modifiedform which optionally is different from the modification that isintroduced by the first process.

In case of a fermentative production of an mRNA in prokaryotes with orwithout a poly(A) tail region it is also possible to include a poly(A)polymerase addition reaction in order to provide such poly(A) tail or toextend an existing poly(A) tail region. In such embodiment, includingmodified adenine nucleosides or adenine nucleotide prodrugs for thereaction in the feeding medium is a preferred option. Alternatively,modified nucleoside triphosphates for the mRNA fermentation process canbe internalized directly using either expression of nucleotidetransporter proteins (D. A. Malyshev, K. Dhami, T. Lavergne, T. Chen, N.Dai, J. M. Foster, I. R. Correa, Jr., F. E. Romesberg, Nature 2014, 509,385-388.) or by adding artificial molecular transporters in the feedingmedium (Zbigniew Zawada et al., Angew. Chem. Int. Ed. 2018, 57,9891-9895).

The processes for producing the mRNA of the invention can be performedusing only one type of modified nucleotide or including one or morenucleotides comprising desired alkyne- or azide-modification. Within thecontext of the present invention, it is preferred to include one or twotypes of equally modified nucleotide, most preferably alkyne- orazide-modified uracil or adenine. As far as the alkyne-modification isconcerned, it is most preferable to include an ethynyl group which, dueto its size, is least prone to negatively affect the transcriptionreaction.

In another preferred embodiment of the invention, two differentlymodified nucleotides are included with the nucleotide mixture duringtranscription. Such a process results in a modified mRNA molecule whichcontains an alkyne- as well as an azide-modification.

No particular restrictions have been observed concerning the amount ofmodified nucleotides to be included during transcription or fermentationor via poly(A) polymerase reaction. Theoretically, all nucleotidesemployed in the in vitro transcription can be modified to containalkyne- or azide-modified nucleobases. It is, however, preferred to useone or two types of modified nucleotides and also to include suchnucleotides in modified as well as in non-modified form. Depending onthe desired modification rate, it is preferred to include modifiedversus non-modified forms of the various nucleotides in a ratio of 1:100to 10:1, preferably 1:10 to 10:1 and further preferably 1:4 to 4:1 or1:2 to 2:1. Most preferably, only one type of modified nucleotide isemployed which can be present in modified form only, or in combinationwith the non-modified form in the above-mentioned ratios. Preferably, a1:1, 1:2 or 1:10 combination of modified to non-modified nucleotide isprovided.

The ratios for introduction of modified nucleotides correspond with thenumber of modifications present in the inventive mRNA. Accordingly, theratio of modified to non-modified nucleosides within the mRNA or thevarious parts, i.e. the UTRs and the ORF, or the UTRs, the ORF and thepoly(A)tail, or the poly(A) tail alone is also preferably 1:100 to 10:1,more preferably 1:10 to 10:1 and further preferably 1:4 to 4:1 or 1:2 or2:1, as well as most preferably 1:1, 1:2 or 1:10.

It is further possible and can be desirable to include differentlymodified natural nucleotides, e.g. pseudouridine orN1-methyl-pseudouridine and/or artificial nucleotides or nucleotidederivatives to improve mRNA stability and enhance translation of theproduced mRNA. More information with regard to differently modifiednucleotides and their incorporation into mRNA during in vitrotranscription can be derived from Svitkin, Y. V. et al., Nucleic AcidsResearch 2017, Vol. 45, No. 10, 6023-6036.

Modified mRNA of the invention which is produced by in vitro mRNAtranscription, by poly(A) polymerase addition reaction on an existingmRNA of interest, by a fermentation process or even completelysynthetically and which comprises at least one of an alkyne- orazide-modification can further be modified via a click reaction toincorporate other molecules of interest, especially labels and/orfunctional molecules as already explained above. For example, detectablelabels like e.g. fluorescent or colored molecules or non-coloredmolecules as mentioned earlier can be introduced. Also, as explainedabove, providing a modified mRNA to produce a fusion protein including,e.g., GFP or eGFP is another preferred option to include a detectablesignal. As a consequence, e.g. delivery and/or expression of thegenerated mRNA can be monitored using fluorescent microscopy, FACS orother detection methods, especially in cell culture experiments. Itsurprisingly has been observed that even relatively big modifications ofthe bases within the ORF are accepted during translation of mRNA at theribosome. For example, Cyanine 5 modified eGFP (enhanced greenfluorescent protein) mRNA is commercially available (TRILINKbiotechnologies, product code LL7701) that contains Cyanine 5 (Cy5)modified uridines. Such mRNA is readily translated to a functionalprotein in cell culture.

Selective modification of solely the poly(A) tail region can be achievedas described above, when poly(A) polymerase adds azide- oralkyne-modified ATP or ATP derivatives to the mRNA. Subsequent clicklabeling of this modified poly(A) tail has only minor effects ontranslation (as the sequence is not translated) and can be used, e.g.for tissue-specific ligands that mediate targeted uptake of the mRNA orto increase mRNA stability against nuclease degradation, as explainedabove. Especially in cases in which it is desired to attach very largemolecules or molecules that due to other reasons impair translation,coupling via the poly(A) tail can be a preferred or even a mandatoryapproach.

The click reaction is well known to the skilled person and it isgenerally referred to Sharpless et al. and Meldal et al., mentionedsupra. The overall conditions for the click reaction are described inthese documents and it is further referred to disclosure in Himo F. etal., J. Am. Chem. Soc., 2005, 127, 210-216, which relates to thepreferred copper-catalyzed azide-alkyne cycloaddition (CuAAC). It isalso referred to EP 2 416 878 B1 with regard to the conditions andreactants for the click reaction as well as to EP 17 194 093, wherein apreferred method for coupling a first molecule to a second molecule in aclick ligation reaction is described. In this context, thecopper-catalyzed click reaction is preferably performed in the presenceof divalent metal cations in the reaction mixture, most preferably inthe presence of Mg²⁺.

While the above-mentioned documents describe click reactions in thecontext of ligating DNA molecules, in general, the same conditions canbe applied within the context of the present invention. Thus, the clickreaction is preferably carried out in the presence of a heterogeneous Cu(I) catalyst. Further, it is preferred to include a Cu (I) stabilizingligand and/or organic solvents, especially DMSO to improve theefficiency of the click reaction, and/or divalent cations (e.g. asdisclosed in PCT/EP2018/076495).

In a further preferred embodiment, the click reaction is performed as astrain-promoted azide-alkyne cycloaddition reaction (SPAAC) as describedearlier with regard to the modified mRNA of the invention. The exactconditions of a CuAAC or a SPAAC reaction can be adapted to theindividual circumstances as long as the basic requirements that areknown to the skilled person are observed. As mentioned above, SPAAC canalso be performed inside of cells. Introducing an alkyne- orazide-modified label into such cells can be useful in order to, aftertransfection of a modified mRNA into cells, monitor e.g. the location ofthe mRNA in the cell.

The present invention allows to produce in a modular and highlyefficient manner modified mRNA molecules which contain modificationswhich impart a stabilizing effect on the mRNA. The modifications arealso useful as anchor molecules to which other substances and moleculescan be linked via a click reaction. Such click reaction is preferablyperformed downstream and separately from the transcription reactionwhich is a tremendous advantage, especially where large and bulkymolecules of interest are to be ligated to the mRNA, which wouldcompletely disrupt the transcription reaction.

Within the context of the present invention, it can be sufficient thatonly a small set of alkyne- and/or azide-modified nucleotides areincorporated during in vitro mRNA production to allow synthesis of awhole range of densely modified mRNAs. This allows for a fastpreparation and screening of many modifications. Moreover, the mRNAs ofthe present invention and processes for producing same permit toincorporate functional groups that are not readily or not at allaccepted by the RNA polymerases during mRNA production but are easilyattached via click reaction after transcription. Incorporation of suchfunctional groups cannot be effected by conventional methods.

Thus, the inventive mRNAs and the processes for their production for thefirst time provide an easy and reliable method to produce stabilized andcustomarily modified mRNAs which can be labelled to follow-up on theiruptake for example in ex vivo cell transfection and can also be modifiedto provide improved cell- or tissue-specific targeting for specific usesin therapy or vaccine preparation.

One preferred application of the mRNAs of the present invention lies intransfection of target cells ex vivo. As mentioned in the backgroundsection, mRNA formulations which are applied systemically and especiallyintravenously are taken up mainly by liver cells, whereas very oftencells of the immune system are the preferred target in order to evoke animmune stimulatory effect or when mRNA is used for direct vaccination.In case that it is desired to incorporate the modified mRNA of theinvention into specific cell types, such cells can be isolated from apatient, especially from a patient's blood, and mRNA transfection can beperformed ex vivo.

A further subject of the present invention, therefore, is a cellpreparation and especially a preparation of cells of the immune system,which includes a modified mRNA of the present invention and is obtainedby ex vivo transfection of cells. In principle, the modified mRNA of thepresent invention can be used to transfect any kind of cell, human,animal or also plant cells. In one aspect of the invention, the cells ofthe cell preparation are of animal or human origin.

This aspect of the invention relates to inter alia adoptive celltransfer (ACT) and its manifold applications and uses which have beendeveloped within the last decades. Autologous as well as non-autologouscells can be treated in order to for example improve immunefunctionality and other characteristics. Preferably, cells of the immunesystem are obtained from a patient and engineered to produce autologousimmune cell which have been proven valuable in treating various diseasesincluding cancer, e.g. B-cell lymphoma. The CAR-T cell based therapy isone such approach in which T-cells are genetically engineered to producechimeric antibody receptors on their surface which recognize and attachto a specific protein or antigen on tumor cells.

The cell preparations of the present invention can be used in the samecontext. Depending on the modified mRNA which is introduced into cells,ensuing expression of a protein can provide for a multitude of effectsof such cells after (re-)application to a patient. The present cellpreparations, accordingly, are not restricted to a small number ofapplications but rather can be considered a vehicle for expression ofmRNA in vivo after (re)application of cells which then produce a proteinof interest and/or exert a certain effect (e.g. immune stimulating ortolerogenic) in the patient due to expression of the protein.

Methods for cell transfection are known to the skilled person and can beadapted to the particular cell type of interest. As an example for suchprocess, it is referred to Moffett et al., mentioned supra. In thecontext of ex vivo transfection it is especially preferred to use anmRNA of the invention which is modified via click reaction to contain acell-specific targeting group which facilitates uptake of the mRNA intothe cell without transfection agents, since immune-cells are especiallydamageable by some of these transfection agent components.

In addition to ex vivo transfection of cells and administering suchtransfected cells to a patient, the modified mRNA of the presentinvention can also be applied directly to a patient. Both cases areconsidered a therapeutic (or also prophylactic) treatment. A furthersubject-matter of the present invention therefore is a pharmaceuticalcomposition which comprises a modified mRNA or a cell preparation of thepresent invention as an active agent. As already mentioned, mRNA basedtherapeutics have recently become important research subjects. A largenumber of applications for mRNA as therapeutic agents has been described(e.g. Sahin et al, Schlake et al. and Kranz et al, all mentioned supra)and the modified mRNA of the present invention can not only be used inall such applications but can even provide for advantages andimprovements thereto. Based on the enhanced stability of the modifiedmRNA and further based on an optionally present functional group,various problems can be solved. An enhanced stability accounts for e.g.a prolonged translation into protein compared to a non-modified mRNA.Further, the presence of a tissue- or cell-targeting group allows fortargeted administration and high specificity of a therapeutic orimmunogenic treatment.

Among suitable applications of the modified mRNAs, the cell preparationsand the pharmaceutical compositions containing such mRNA or cellpreparation are gene or protein replacement therapy, targeted transientgene delivery and genome engineering/gene editing (e.g. mRNA coding fora targeting endonuclease and a guide RNA like in the CRISPR/Cas9 systemor similar), infectious disease vaccination, cancer immunotherapy, aswell as cell-specific gene expression for a treatment of inheriteddiseases.

Gene replacement therapy can be considered for the treatment of a largenumber of diseases. For many diseases for which a deficiency ormalfunction in a protein or enzyme is a leading cause or consequence,administration of the required active protein to the patient isessential to avoid immediate or consequential damage. However,continuous administration of proteins can cause intolerance or othernegative side effects.

Furthermore, in order to provide sufficient amounts of a certain proteinto a patient, high concentrations of such proteins need to beadministered, sometimes as high as 100 mg/ml and up to 20 g of theprotein per day and patient. As a further problem in protein replacementtherapy, it can also be difficult to administer the protein into cells.On the other hand, it could be shown that for some mRNAs it issufficient to provide 50 to 100 μg per dose to achieve a sufficientlyhigh intracellular protein level in patients. Thus, the presentinvention and especially the possibility to target specific cells ortissues, provides a convenient solution to problems encountered withcurrent protein replacement therapy approaches. By using modified mRNAsof the present invention in protein replacement therapy, it is forexample in many cases sufficient to inject the mRNA whereas currentprotein replacement therapies require infusions which usually are timeconsuming and physically demanding for the patient.

Examples of diseases requiring protein replacement or at least constantprotein supplementation include protein deficiency diseases, manymetabolic diseases like type I diabetes, and also inherited disorders,especially lysosomal storage diseases like Morbus Gaucher or MorbusHunter.

In the context of gene replacement therapy, including the modified mRNAinto cells ex vivo as well as in vivo can be considered. Especially if acell- or tissue-specific targeting-group is included with the mRNA ofthe invention, even in vivo insertion of the modified mRNA into targetcells is expected to be highly efficient. The present invention allowsfor an endogenous translation of mRNA into protein, thus, avoidingadverse effects as mentioned above. Nevertheless, also ex vivo insertionof the mRNA into target cells and (re)administration of such targetcells into patients is a further option within the context of thepresent invention, as mentioned above.

The pharmaceutical composition according to the invention can also beapplied as mRNA vaccine. Vaccination is effected based on in situprotein expression to induce an immune response. Since any protein canbe expressed from the modified mRNA of the present invention, thepresent pharmaceutical compositions can offer maximum flexibility asregards the desired immune response. Using the modified mRNA alsoprovides a very fast immunization alternative compared to conventionalmethods for which it is necessary to produce various proteinconstituents or even inactivated viral particles. Conventional methodsusually require performing different production processes whereas usingthe present invention, various mRNAs encoding different proteins orprotein parts relating to the infectious agent can be produced in thesame preparation process. Immunization by mRNA vaccination can even beachieved by single vaccinations and using only low mRNA doses. Asopposed to DNA vaccines, RNA vaccines do not need to cross the nuclearenvelope, but it rather is sufficient for them to reach the cellcytoplasm by crossing the plasma membrane. Further information regardingthe development of mRNA vaccines is disclosed e.g. in Schlake et al.,mentioned supra, and is applicable also in the context of the presentinvention. The pharmaceutical composition including the modified mRNA ofthe invention can be employed as prophylactic as well as therapeuticvaccines. The vaccines can be directed against any kind of pathogens,e.g. viruses like Zika virus which recently has become a major focus ofattention (Pardi et al, mentioned supra).

In addition to a vaccination against exogenous pathogens, thepharmaceutical compositions of the present invention can also be used asanti-tumor vaccines or to stimulate the immune system within the contextof cancer immunotherapy. Especially systemic RNA delivery to dendriticcells or macrophages offers the possibility to exploit antiviral defensemechanisms for cancer immunotherapy as described by Kranz et al.,mentioned supra. Targeting e.g. macrophages or dendritic cells with anmRNA expressing a protein specific to or within the context of a certaintype of cancer leads to presentation of parts of such protein by MHCmolecules and elicits a potent and specific immune response.Accordingly, the modified mRNA of the present invention can also be usedin the context of antigen-encoding mRNA pharmacology.

In the context of RNA-based immunotherapy and vaccination, it isespecially preferred to include an (m)RNA adjuvant as described above inan immunostimulatory pharmaceutical composition. The adjuvant providesfor a stimulation of the innate immune response and thus furtherenhances the immunotherapeutic effect. In this context, the modifiedmRNA of the present invention and the inventive (m)RNA adjuvant can havethe same or a similar sequence and even encode the same protein. On theother hand, also a non-coding (m)RNA adjuvant or an (m)RNA adjuvantencoding a different protein or peptide can be combined to achieve thedesired adjuvant effect.

Targeted gene editing using specific endonucleases and guide RNAs is afurther application of the modified mRNA or pharmaceutical compositionof the present invention.

The recently developed, ground-breaking CRISPR/Cas9 technology enablesspecific gene editing, allows to introduce, delete or silence genes andis even able to exchange nucleotides within a gene. The method describedby Charpentier and Doudna involves Cas-proteins which areribonucleoproteins and endonucleases which bind to specific chemicallysynthesized CRISPR RNA (crRNA) sequences and cut DNA in the vicinity ofsuch RNA sequences. In order to direct the endonuclease activity to thedesired target DNA sequence, a so-called guide RNA is used which iscomplementary to the target DNA sequence. The guide RNA can take twoforms, either a complex of a long, chemically synthesizedtrans-activating CRISPR RNA (tracrRNA) plus the crRNA, or a synthetic orexpressed single guide RNA (sgRNA) that consist of both the crRNA andtracrRNA as a single construct. The modified mRNA of the presentinvention can be used in this context to encode one or both of theendonuclease and the guide RNA, preferably as a sgRNA which iscomplementary to a specific DNA sequence of interest.

The major advantage of the transient expression of the gene editingendonuclease and guide RNA from mRNA compared to the current technology,is the reduced risk of non-specific gene editing, since the geneexpression of the genetic tool from mRNA is limited to a short period oftime (a few days) and not constantly expressed from an integrated genomeelement.

Gene editing applications have high importance and can be applied in thecontext of a huge variety of therapeutic applications, e.g. genereplacement therapy, cancer therapy and treatment of inherited diseases.Use of the modified mRNA of the invention in the context of all suchapplications is included within the scope of the present invention.

Also for other potential therapeutic applications described for mRNAs inthe prior art or which are developed in the future, use of respectivemRNAs including the modifications as described herein is of advantagedue to their improved stability. Further, including a tissue- orcell-specific targeting molecule to the mRNA via the downstream clickreaction, allows for a more specific and accordingly more efficient usein therapy. Especially any desired protein expression or also anyimmunization reaction can be precisely targeted to the location in apatient where the therapeutic or immunizing effect is required.

A preferred embodiment of the pharmaceutical composition of the presentinvention includes the modified mRNA together with a pharmaceuticallyacceptable carrier, excipient and/or adjuvant, preferably a modified(m)RNA adjuvant as described above containing the same modification asthe modified mRNA and which is complexed with a cationic or polycationiccompound. In a further preferred embodiment, the pharmaceuticalcomposition comprises complexing agents, which protect the mRNA furtherfrom degradation. The complexing agent may improve and enhance uptake bycells and concurrent translation into protein. As complexing agents,lipids or polymers can be included in the pharmaceutical composition. Ina further preferred embodiment, the pharmaceutical composition cancontain the modified mRNA encapsulated in liposomes.

In a further preferred embodiment, the pharmaceutical compositioncontains cationic lipids. Agents that further improve the delivery ofnucleic acids to the cytosols can also be preferably included in thepharmaceutical compositions of the present invention. Such agents can betailored to the specific route of delivery. In summary, thepharmaceutical compositions of the present invention, while includingthe inventive modified mRNA as active agent, can include any furthersubstances for improving further the stability of the active substance,enhancing delivery to the cytoplasm of target cells and providing othercomplementary or synergistic effect.

Within the context of the present invention, a pharmaceuticalcomposition is included which as an active agent contains a cellpreparation, especially a preparation of cells of the immune system,which is obtained by ex vivo transfection of cells with a modified mRNAof the present invention. The transfected cells can be returned to thepatients in order to benefit from the effects of the modified RNAincluded in the cells. Also for this preferred embodiment of apharmaceutical composition of the invention, pharmaceutically acceptableadjuvants or excipients or carriers can be included as outlined before.

A further subject of the present invention is a diagnostic compositioncontaining a modified mRNA of the present invention or a celltransformed with a modified mRNA of the present invention for in vitroor in vivo screening for the presence, delivery and/or distribution ofthe inventive mRNA in cells, tissues or organs. For such purpose,preferably the modified mRNA already includes a detectable label whichwas introduced via a click reaction. Such label preferably is afluorophore or a radionuclide, preferably a positron emittingradionuclide. Detecting and possibly also quantifying the detectablelabel allows to observe and detect delivery of the modified mRNA tocells, tissues or organs, the distribution therein or to monitor there-administration of cells into a patient. Accordingly, a diagnosticcomposition of the present invention contains a modified mRNA of theinvention, preferably an mRNA containing at least one detectable label.Within this context, including a modified mRNA which upon expressionproduces a detectable protein, e.g. a fusion protein including afluorescent protein, is a further preferred embodiment.

In a further aspect of the present invention and as mentioned earlier,also plant cells can be transfected using the modified mRNA of thepresent invention. Such transfected plant cells are also encompassedwithin the scope of the present invention. The modified mRNA can beincluded e.g. in order to introduce genetic information, especially fortransient expression of certain proteins in such plant cells, or foranalytic or diagnostic purposes e.g. as labeled probes. Conferringdisease or pest resistance or tolerance are only some examples ofpossible applications and beneficial effects of introducing inventivemRNAs into plant cells or plants. The use of a modified mRNA of thepresent invention for transfection of plant cells or plants,accordingly, is also a subject of the present invention.

Still a further subject of the present invention is a kit for preparinga modified mRNA of the present invention. Such kit contains the varioussubstances required for preparing the modified mRNA via in vitrotranscription, a fermentation process or a poly(A) polymerase additionreaction, i.e. an RNA polymerase and/or poly(A) polymerase, alkyne- orazide-modified nucleotides as well as unmodified nucleotides andoptionally further buffer substances and solvents or further substancesrequired for the process. In preferred embodiments, also alkyne- and/orazide-modified detectable labels or functional molecules as well assubstances required for performing the click reaction between themodified mRNA and the labels or functional molecules are included. Also,a kit for producing the modified mRNA of the present invention entirelysynthetically is a further subject of the present invention. Therequired substances can be provided in separate containers or can becombined as far as no adverse reaction occurs between such combinedsubstances. As regards the various substances to be included in such kitof parts, it is referred to the above description regarding the modifiedmRNA of the invention and the processes for preparing such modifiedmRNA. As far as production of the modified (m)RNA adjuvant is concerned,such kit preferably also contains a cationic or polycationic compoundwhich according to a preferred embodiment is used to form a complex withthe (m)RNA. The kits according to the present invention can also containfurther substances facilitating the delivery of the modified mRNA of theinvention to cells ex vivo or in vivo.

In preferred embodiments, a kit includes at least one modified mRNA ofthe invention, preferably containing a detectable label or a functionalmolecule introduced via click reaction, or it provides the modified mRNAand the alkyne- or azide-modified label and/or functional molecule andoptionally other click reagents in separate containers.

In a further embodiment of the invention, a kit for delivery of themodified mRNA to a patient contains an mRNA and preferably also an(m)RNA adjuvant, both modified according to the present invention. Themodified mRNA and the modified (m)RNA adjuvant can be contained in onesingle container or in separate containers and both can optionallyinclude an alkyne- or azide-modified label or functional molecule eitheralready attached via click reaction or in separate containers forsubsequently performing the click reaction. The kit can further containother pharmaceutically acceptable carriers and adjuvants, again eitherin separate containers or combined with at least one other constituentof the kit.

It will be apparent to the skilled person that many differentcombinations of substances can be included in kits which facilitate thepreparation or the use of the modified mRNA of the invention. Allembodiments and variations thereof described above in the context of thepresent invention are also applicable for the kits described here. Allsuitable combinations of substances are included for the purposes of thepresent invention.

A further subject of the present invention relates to a method forstabilizing RNA, especially mRNA, such method including introducing analkyne- and/or an azide-modification by including at least one of thefour standard types of nucleotides (ATP, CTP, GTP and UTP) in partly orcompletely alkyne- and/or azide-modified form during RNA synthesisand/or in a poly(A) polymerase addition reaction to produce a modified(mRNA). As described above, the modification of an RNA, especially anmRNA by including alkyne- and/or azide-modified nucleotides, andespecially by optionally including also one or more of a detectablelabel and a functional molecule via a click reaction, results in astabilizing effect on the RNA molecules. Thus, a corresponding method isconsidered a further important aspect included with the presentinvention.

Still a further subject of the present invention is an in vitro methodfor qualitatively or quantitatively determining the presence and/orexpression of an mRNA according to the present invention in targetcells. In this context, the transfection efficiency, a quantification ofmRNA delivery and expression can be determined at a single cellresolution via FACS analysis. Fluorescence-activated cellsorting/scanning, FACS, is well-known to the skilled person. Thefluorescent labels that can be introduced into the modified mRNA of thepresent invention via click-reaction can be determined based on thismethod. Furthermore, mRNA delivery and expression of the encoded proteincan be detected using a fluorescent protein, e.g., the GFP protein oreGFP protein which in a preferred embodiment can be co-expressed with aprotein of interest as a fusion protein or as two separate proteins, asdescribed above.

Accordingly, using the FACS method, the influence of the modification oncell transfection and the expression level of the protein encoded by themodified mRNA of the invention can easily be determined. Also studyingeffects of different labels on the expression level can be performed viaFACS analysis.

In such FACS analysis, e.g. a comparison of non-transfected cells andtransfected cells allows to detect fluorescent signals which can beattributed to the fluorescent label included in the modified mRNA of theinvention or the fluorescent protein expressed by translation of themodified mRNA of the invention. Also a comparison of transfectionreactions of the same target cells with modified mRNA of the inventionversus non-modified mRNA having the same nucleotide sequence can ensurethat the modification per se does not negatively influence thetransfection efficiency.

All information disclosed above with regard to one subject of thepresent invention is considered to equally apply in the context of othersubjects for which this information, even if not explicitly repeated,has recognizable relevance within the context of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a general scheme of modified mRNA production andapplication. Using e.g. 5-ethynyl UTP (EUTP) it is possible to insertalkyne groups available for click reaction with the 5′-UTR, 3′-UTR andthe ORF. Selective labelling of the poly(A) tail is possible using e.g.7-ethynyl 7-deaza ATP and a poly(A) polymerase.

FIG. 2 shows the general schematic production of alkyne-modified mRNAusing e.g. T7 RNA polymerase and a nucleotide mixture including EUTP(structural formula).

FIG. 3 shows the results of transfection of non-alkyne (A), alkyne (B)and dye (C) modified mRNA coding for eGFP into HeLa cells.

FIG. 4 shows the general schematic production of alkyne-modified mRNAusing e.g. poly(A) polymerase and the alkyne modified nucleotide EATP(structural formula).

FIG. 5 shows the results of transfection of Eterneon-red 645 modifiedmRNA (alkyne modification in poly(A) tail only) coding for eGFP intoHeLa cells.

FIG. 6 shows a map and the complete sequence (from T7 promoter topoly(A) end) of the plasmid used in linearized form as DNA templateduring the T7 RNA polymerase reaction in the Examples. The sequence isalso referred to as SEQ ID NO: 2.

FIG. 7 shows the result of experiments to prove incorporation of EATPinto the poly(A) tail of an RNA as described in Example 3.

FIG. 8 shows a general scheme of the production of site-specificazide-modified mRNA (single azide at the end of the poly(A) tail only)using yeast poly(A) polymerase and the azide-modified nucleotide AzddATPas described in Example 4.

FIG. 9 shows a transfection of 3′-poly(A) tail Cy3 modified mRNA codingfor eGFP into HeLa cells. After 24 h incubation at 37° C. greenfluorescence of the eGFP was observed (eGFP filter). For the Cy3 labeledmRNA the localization of the mRNA was observed using Cy3 filtersettings.

FIG. 10 shows a general scheme for production of double labeledazide/alkyne modifed mRNA (internal alkyne groups using T7 RNApolymerase and EUTP, one terminal azide at 3″end using AzddATP and yeastpoly(A) polymerase), as in Example 5.

FIG. 11 shows the transfection of internal Eterneon Red modified and3′-poly(A) tail Cy3 modified mRNA coding for eGFP into HeLa cells. After24 h incubation at 37° C. green fluorescence of the eGFP was observed(eGFP filter). For the Cy3 labeled mRNA the localization of the mRNA wasobserved using Cy3 filter settings, for the Eterneon Red labeled mRNAthe localization of the mRNA was observed using Cy5 filter settings.

FIGS. 12 to 16 show the results of FACS analyses for untransfected HeLacells (FIG. 12), HeLa cells transfected with non-modified mRNA encodingeGFP (FIG. 13), HeLa cells transfected with alkyne-modified mRNAencoding eGFP (FIG. 14) and Eterneon Red-/alkyne-modified mRNA encodingeGFP (FIGS. 15 and 16) and allow quantification of protein expressiondepending on modification and uptake of dye-labeled mRNA (FIGS. 15 and16).

FIG. 17 shows a schematic representation of one embodiment of theinvention: Bacterial cells are feeded with, e.g., 5-ethynyluridine andtransformed with a plasmid containing the sequence necessary for theproduction of the mRNA. The newly synthetized mRNA containing EU is thenpurified by poly(T) resin and/or beads having poly(T) oligonucleotidesattached.

The following examples further illustrate the invention:

EXAMPLES Example 1

Alkyne-modified mRNA coding for the enhanced green fluorescent protein(eGFP) was produced by in vitro transcription (IVT) from a DNA templateusing T7 RNA polymerase and nucleotide mixtures. Here5-ethynyl-uridine-5′-triphosphate (EUTP) was included in the nucleotidemixture to generate an alkyne-modified mRNA according to FIG. 2 forsubsequent transfection into Henrietta Lacks' immortal cells (HeLacells). The generated mRNA contains a 5′-cap, untranslated regions(UTR), the protein coding part (open reading frame, ORF) and a poly(A)tail.

mRNA Production

In a 50 μL reaction volume 20 units of T7 RNA polymerase, 1 μg oftemplate DNA and several nucleotides were combined in transcriptionbuffer (40 mM Tris-HCl, pH 7.9, 6 mM MgCl₂, 4 mM spermidine, 10 mM DTT).

A) Final nucleotide concentrations for non-alkyne modified mRNAproduction were: 1.0 mM GTP, 4.0 mM A.R.C.A.(P1-(5′-(3′-O-methyl)-7-methyl-guanosyl) P3-(5′-(guanosyl))triphosphate,Cap Analog), 1.25 mM CTP, 1.25 mM UTP, 1.25 mM ψUTP (pseudouridinetriphosphate), 1.5 mM ATP.

B) Final nucleotide concentrations for alkyne modified mRNA productionwere: 1.0 mM GTP, 4.0 mM A.R.C.A.(P1-(5′-(3′-O-methyl)-7-methyl-guanosyl) P3-(5′-(guanosyl))triphosphate,Cap Analog), 1.25 mM CTP, 1.25 mM EUTP (5-ethynyluridine triphosphate),1.25 mM ipUTP (pseudouridine triphosphate), 1.5 mM ATP.

C) Final nucleotide concentrations for alkyne modified mRNA productionand subsequent click labeling were:

1.0 mM GTP, 4.0 mM A.R.C.A. (P1-(5′-(3′-O-methyl)-7-methyl-guanosyl)P3-(5′-(guanosyl))triphosphate, Cap Analog), 1.25 mM CTP, 0.625 mM EUTP(5-ethynyluridine triphosphate), 0.625 mM ψUTP (pseudouridinetriphosphate), 0.625 mM UTP, 1.5 mM ATP.

The mixture was incubated for 2 hours at 37° C. and then 2 units ofDNAse I were added and incubated for 15 minutes at 37° C. The mRNA waspurified by a spin column method according to manufacturers' instructionfor PCR products (PCR purification kit from Qiagen). This yielded 13.3μg of mRNA for A, 12.3 μg B and 14.3 μg for C, which was directly usedfor transfection when no click labeling was needed (A and B). When clicklabeling was performed (C), 2 μg of RNA, 1 nmol Eterneon Red 645 Azide(baseclick GmbH), a single reactor pellet and 0.7 μL 10× Activator²(baseclick GmbH, Oligo² Click Kit) were combined in a total reactionvolume of 7 μL. The reaction mixture was incubated at 45° C. for 30 minand then cleaned using a spin column method according to manufacturers'instruction for PCR products (PCR purification kit from Qiagen).

For transfection a commercial kit (jetMESSENGER™ from POLYPLUSTRANSFECTION®) was used according to manufacturers' instructions using0.5 μg of mRNA and 25,000 HeLa cells (CLS GMBH) reaching confluence. Thecells were incubated at 37° C. for 24 hours before analysis under thefluorescent microscope, GFP filter: (470/22 excitation; 510/42 emission)and Cy5 filter (628/40 excitation; 692/40 emission) were used.

FIG. 3 shows the results of transfection of non-alkyne (A), alkyne (B)and dye (C) modified mRNA coding for eGFP into HeLa cells. After 24 hincubation at 37° C. green fluorescence of the eGFP was observed (GFPfilter). For the Eterneon Red labeled mRNA (C) the localization of themRNA was observed using Cy5 filter settings.

In the bright field image cell morphology of healthy HeLa cells wasobserved (FIG. 3, A-C), using the GFP filter protein expression of theeGFP was visible (exposure time 120 ms for A-B, 250 ms for C). For theclick labeled mRNA (FIG. 3, C) also the localization of the mRNA wasobserved using the Cy5 filter settings of the microscope.

Supporting Information:

Structure of the modified nucleotides used during the T7 RNA polymerasereaction described above.

The map and complete sequence (from T7 promoter to poly(A) end) of theplasmid used in a linearized form as DNA template during the T7 RNApolymerase reaction described above is shown in FIG. 6. The sequence isalso referred to SEQ ID NO: 2.

Example 2

Alkyne-modified mRNA coding for the enhanced green fluorescent protein(eGFP) was produced by in vitro transcription (IVT) from a DNA template(FIG. 6) using T7 RNA polymerase and nucleotide mixtures. Here7-ethynyl-adenine-5′-triphosphate (EATP) was incorporated in the IVTmRNA after the T7 RNA polymerase reaction by poly(A) polymerase togenerate an alkyne-modified mRNA according to FIG. 4 for subsequentclick labeling and transfection into Henrietta Lacks' immortal cells(HeLa cells). The generated mRNA contains a 5′-cap, untranslated regions(UTR), the protein coding part (open reading frame, ORF) and a poly(A)tail alkyne labeled.

mRNA Production

In a 50 μL reaction volume 20 units of T7 RNA polymerase, 1 μg oflinearized template DNA and several nucleotides were combined intranscription buffer (40 mM Tris-HCl, pH 7.9, 6 mM MgCl₂, 4 mMspermidine, 10 mM DTT).

Final nucleotide concentrations for non-alkyne modified mRNA productionwere: 1.0 mM GTP, 4.0 mM A.R.C.A.(P1-(5′-(3′-O-methyl)-7-methyl-guanosyl) P3-(5′-(guanosyl))triphosphate,Cap Analog), 1.25 mM CTP, 1.25 mM UTP, 1.25 mM ψUTP (pseudouridinetriphosphate), 1.5 mM ATP.

The mixture was incubated for 2 hours at 37° C. and then 2 units ofDNAse I were added and incubated for 15 minutes at 37° C. The mRNA waspurified by a spin column method according to manufacturers' instructionfor PCR products (PCR purification kit from Qiagen). This yielded 12.3μg of mRNA which was directly used for poly(A) polymerase reaction withEATP.

In a 20 μL reaction volume 5 units of E. coli poly(A) polymerase, 4.2 μgof mRNA prepared before and a solution of 1 mM EATP were combined inreaction buffer (250 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl₂, pH 7.9)

The mixture was incubated for 1 hour at 37° C. The mRNA was purified bya spin column method according to manufacturers' instruction for PCRproducts (PCR purification kit from Qiagen). This yielded 4 μg of mRNA.

The click labeling was performed using 1.1 μg of RNA, 1 nmol EterneonRed 645 Azide (baseclick GmbH), a single reactor pellet and 0.7 μL 10×Activator² (baseclick GmbH, Oligo² Click Kit) were combined in a totalreaction volume of 7 μL. The reaction mixture was incubated at 45° C.for 30 min and then cleaned using a spin column method according tomanufacturers' instruction for PCR products (PCR purification kit fromQiagen).

For transfection a commercial kit (jetMESSENGER™ from POLYPLUSTRANSFECTION®) was used according to manufacturers' instructions using0.5 μg of mRNA and 25,000 HeLa cells (CLS GMBH) reaching confluence. Thecells were incubated at 37° C. for 24 hours before analysis under thefluorescent microscope, GFP filter: (470/22 excitation; 510/42 emission)and Cy5 filter (628/40 excitation; 692/40 emission) were used.

FIG. 5 shows the results of transfection of Eterneon Red modified mRNAcoding for eGFP into HeLa cells. After 24 h incubation at 37° C. greenfluorescence of the eGFP was observed (GFP filter). For the Eterneon Redlabeled mRNA the localization of the mRNA was observed using Cy5 filtersettings.

In the bright field image cell morphology of healthy HeLa cells wasobserved (FIG. 5), using the GFP filter protein expression of the eGFPwas visible (exposure time 120 ms). Localization of the mRNA labeledwith Et-Red was observed using the Cy5 filter settings of themicroscope.

Example 3

In order to prove incorporation of the EATP(Ethynyl-adenosine-5′-triphosphate) within the poly(A) tail a short RNAoligonucleotide (31 mer, CUAGUGCAGUACAUGUAAUCGACCAGAUCAA, SEQ ID NO: 1)was used as template for the poly(A) polymerase reaction using:

A) 1 mM ATP

B) 1 mM EATP;

C) 0.5 mM ATP and 0.5 mM EATP.

In a 20 μL reaction volume 5 units of Escherichia coli poly(A)polymerase, 2 μg of RNA (31 mer) and nucleotide (final concentration ofA-C) were combined in reaction buffer (250 mM NaCl, 50 mM Tris-HCl, 10mM MgCl₂, pH 7.9). The mixtures were incubated for 30 minutes at 37° C.or for 16 hours at 37° C.

The results were analyzed by denaturing polyacrylamide gelelectrophoresis (7 M urea, lx TBE, 7% polyacrylamide gel, constantvoltage 100 V, 1 h). Compared to the template RNA oligonucleotide (FIG.7, Lane 2) a band or smear at higher molecular weight appeared for allsamples, which were incubated in the presence of the poly(A) polymeraseusing different nucleotides and incubation durations (FIG. 7, Lane 3-7).This indicated successful incorporation of ATP or its alkyne analogEATP. Within 30 min incubation the incorporation of ATP (FIG. 7, Lane 3)was more efficient compared to EATP (FIG. 7, Lane 4) or a mixture ofEATP and ATP (FIG. 7, Lane 5). By extending the incubation time for theincorporation of EATP to 16 h, the length of the poly-EA-addition wasincreased (FIG. 7, Lane 6) in comparison to 30 min incubation (FIG. 7,Lane 4). Interestingly, for the nucleotide mixture containing ATP andEATP no change was observed after 16 h (FIG. 7, Lane 7) compared to 30min.

FIG. 7 shows the ethidium bromide stained 7% denaturing polyacrylamidegel of different polyadenylation reactions as described above. In eachlane 500 ng of RNA were loaded. Lane 1: low molecular weight DNA ladder(New England Biolabs), Lane 2: 31 mer RNA oligonucleotide template, Lane3: polyadenylation reaction with 1 mM ATP for 30 min, Lane 4: like 3 but1 mM EATP, Lane 5: like 3 but 0.5 mM EATP and 0.5 mM ATP, Lane 6: like 4but 16 h incubation, Lane 7: like 5 but 16 h incubation.

Example 4

Azide-modified mRNA coding for the enhanced green fluorescent protein(eGFP) was produced by in vitro transcription (IVT) from a DNA templateusing T7 RNA polymerase and nucleotide mixture. Here3′-azido-2′,3′-dideoxyadenosine (AzddATP) was incorporated, thusterminating the elongation, in the IVT mRNA after T7 RNA polymerasereaction using yeast poly(A) polymerase to generate a site-specificsingle azide modified mRNA according to FIG. 8 for subsequenttransfection in Henrietta Lacks' immortal cells (HeLa cells). Thegenerated mRNA contains a 5″-cap, untranslated regions (UTR). Theprotein coding part (open reading frame, ORF) and a poly(A)-tail with asingle terminal azide.

mRNA Production

In a 50 μL reaction volume 20 units of T7 RNA polymerase, 1 μg oftemplate DNA and several nucleotides were combined in transcriptionbuffer (40 mM Tris-HCl, pH 7.9, 6 mM MgCl₂, 4 mM spermidine, 10 mMdithiothreitol). Final nucleotide concentrations were:

1.0 mM GTP, 4.0 mM A.R.C.A. (P1-(5′-(3′-O-methyl)-7-methyl-guanosyl)P3-(5′-(guanosyl)) triphosphate, cap analog), 1.25 mM CTP, 1.25 mM UTP,1.25 mM ipUTP (pseudouridine triphosphate), 1.5 mM ATP.

The mixture was incubated for 2 hours at 37° C. and then 2 units ofDNAse I were added and incubated for 15 minutes at 37° C. The mRNA waspurified by a spin column method according to manufacturers' instructionfor PCR products (PCR purification kit from Qiagen). This yielded 13.7μg of mRNA which was directly used for yeast poly(A) addition with theazide-containing ATP analog AzddATP.

In a 25 μL reaction volume 600 units of yeast poly(A) polymerase, 5.8 μgof purified IVT mRNA and 0.5 mM AzddATP were combined in reaction buffer(10% (v/v) glycerol, 20 mM Tris-HCl, 0.6 mM MnCl₂, 20 μM EDTA, 0.2 mMDTT, 100 μg/mL acetylated BSA, pH 7.0) and the solution was incubatedfor 20 minutes at 37° C. Modified mRNA was purified by a spin columnmethod according to manufacturers' instruction for PCR products (PCRpurification kit from Qiagen). This yielded 4.8 μg of mRNA.

Click labelling was performed using 4.8 μg of RNA and 2 nmol ofDBCO-sulfo-Cy3 (Jena Bioscience cat. no. CLK-A140-1), combined in atotal reaction volume of 30 μL. The reaction mixture was incubated atroom temperature overnight and then cleaned using a spin column methodaccording to manufacturers' instruction for PCR products (PCRpurification kit from Qiagen). This yielded 4.0 μg of mRNA.

For transfection of modified mRNA a commercial kit (jetMESSENGER™ fromPOLYPLUS TRANSFECTION®) was used according to manufacturers'instructions using 0.5 μg of Cy3 labeled mRNA and 25.000 HeLa cells (CLSGMBH) reaching confluency. The cells were incubated at 37° C. for 24hours before analysis under the fluorescent microscope, GFP filter:(470/22 excitation; 510/42 emission) and Cy3 filter (531/40 excitation;593/40 emission) were used.

In the bright field image cell morphology of healthy HeLa cells wasobserved (FIG. 9 shows a), using the GFP filter protein expression ofthe eGFP was visible (exposure time 120 ms). Localization of the mRNAlabelled with Cy3 was observed using the Cy3 filter settings of themicroscope.

Example 5

Azide/alkyne-modified mRNA coding for the enhanced green fluorescentprotein (eGFP) was produced by in vitro transcription (IVT) from a DNAtemplate using T7 RNA polymerase and nucleotide mixture and yeastpoly(A) polymerase. Here 5-ethynyl-uridine-5′-triphosphate (EUTP) wasincluded in the nucleotide mixture to generate an alkyne-modified mRNAfollowed by incorporation of 3′-azido-2′,3′-dideoxyadenosine (AzddATP),thus terminating the elongation and introducing one single azide. Thisis the first example of dual labelling of the mRNA.

mRNA Production

In a 50 μL reaction volume 20 units of T7 RNA polymerase, 1 μg oftemplate DNA and several nucleotides were combined in transcriptionbuffer (40 mM Tris-HCl, pH 7.9, 6 mM MgCl₂, 4 mM spermidine, 10 mMdithiothreitol). Final nucleotide concentrations were:

1.0 mM GTP, 4.0 mM A.R.C.A. (P1-(5′-(3′-O-methyl)-7-methyl-guanosyl)P3-(5′-(guanosyl))triphosphate, Cap Analog), 1.25 mM CTP, 0.625 mM EUTP(5-ethynyluridine triphosphate), 0.625 mM ψUTP (pseudouridinetriphosphate), 0.625 mM UTP, 1.5 mM ATP.

The mixture was incubated for 2 hours at 37° C. and then 2 units ofDNAse I were added and incubated for 15 minutes at 37° C. The mRNA waspurified by a spin column method according to manufacturers' instructionfor PCR products (PCR purification kit from Qiagen). This yielded 13.9μg of mRNA which was directly used for yeast poly(A) addition with theazide-containing ATP analogue AzddATP.

In a 25 μL reaction volume 600 units of Yeast Poly(A) polymerase, 5.8 μgof purified IVT mRNA and 0.5 mM AzddATP were combined in reaction buffer(10% (v/v) glycerol, 20 mM Tris-HCl, 0.6 mM MnCl₂, 20 μM EDTA, 0.2 mMDTT, 100 μg/mL acetylated BSA, pH 7.0) and the solution was incubatedfor 20 minutes at 37° C. Modified mRNA was purified by a spin columnmethod according to manufacturers' instruction for PCR products (PCRpurification kit from Qiagen). This yielded 4.35 μg of mRNA.

The first click labelling (strain promoted azide-alkyne cyclo-addition,SPAAC) was performed using 4.35 μg of RNA and 2 nmol of DBCO-sulfo-Cy3(Jena Bioscience cat no. CLK-A140-1), combined in a total reactionvolume of 30 μL. The reaction mixture was incubated at room temperatureovernight and then cleaned using a spin column method according tomanufacturers' instruction for PCR products (PCR purification kit fromQiagen). This yielded 2.55 μg of mRNA.

A second click reaction (Cu catalysed azide-alkyne Cyclo-addition,CuAAC) was performed with 2 μg of RNA, 1 nmol Eterneon Red 645 Azide(baseclick GmbH), a single reactor pellet and 0.7 μL 10× Activator²(baseclick GmbH, Oligo² Click Kit) combined in a total reaction volumeof 7 μL. The reaction mixture was incubated at 45° C. for 30 min andthen cleaned using a spin column method according to manufacturers'instruction for PCR products (PCR purification kit from Qiagen).

For transfection a commercial kit (jetMESSENGER™ from POLYPLUSTRANSFECTION®) was used according to manufacturers' instructions using0.5 μg of mRNA and 25,000 HeLa cells (CLS GMBH) reaching confluence. Thecells were incubated at 37° C. for 24 hours before analysis under thefluorescent microscope, GFP filter: (470/22Ex; 510/42 Em), Cy5 filter(628/40Ex; 692/40 Em) and Cy3 filter (531/40 excitation; 593/40emission) were used.

In the bright field image cell morphology of healthy HeLa cells wasobserved (FIG. 11). Using the GFP filter, protein expression of the eGFPwas visible (exposure time 120 ms). Localization of the mRNA labelledwith Cy3 and Eterneon Red was observed using the Cy3 and Cy5 filtersettings of the microscope, proving dual labelling with two differentmolecules.

Example 6: Relative Quantification of mRNA Expression ViaFluorescence-Activated Cell Sorting/Scanning (FACS)

This experiment was intended to evaluate the expression level of invitro transcribed (IVT) eGFP mRNA in cells using a FACS device. eGFPexpression is directly monitored via its fluorescence emission at 509 nmupon excitation at 475 nm and can indicate whether introduction of afunctional group into the RNA, e.g. a terminal alkyne or a dye moleculecan change the expression level. Moreover, uptake of dye-modified mRNAcan be monitored on a second fluorescence channel. Variations in theexpression level within the cell culture population can be detected toevaluate mRNA preparation homogeneity.

Three different IVT mRNAs were prepared by using the T7 RNA polymeraseand a DNA template with different nucleotide mixtures, and if necessarya subsequent click reaction:

-   A) unmodified nucleotides mixture (=unmodified eGFP mRNA),-   B) nucleotide mixture containing 5-ethynyl-uridine 5′-triphosphate    (=alkyne modified eGFP mRNA),-   C) like B) but subsequent click reaction in the presence of    Eterneon-Red azide (Cy5 analog, baseclick GmbH) (=Eterneon Red eGFP    mRNA).

2 μg of each mRNA preparation were used for transfection into HenriettaLacks' immortal cells (HeLa) and buffer without mRNA as a negativecontrol. After 24 h incubation at 37° C. the cells were detached, fixedand then at least 10000 cells were analysed using FACS (FACS Canto II,BECTON DICKINSON).

All samples were analysed using two channels, one for eGFP fluorescenceto evaluate the protein expression and one for the Eterneon Red dyefluorescence to evaluate the presence of dye-labelled mRNA. Thisresulted in a histogram and dot plot which are shown for each experimentand fluorescence channel as reported below. The histogram displays thenumber of counted cells per fluorescence intensity and the dot plotdisplays the cell internal organization (SSC) in correlation to thefluorescence intensity (eGFP or Eterneon Red). Data from 10.000 counts(=10.000 cells) were collected for each sample.

-   a) HeLa cells, which were not transfected with mRNA, were analysed    as a negative control and to establish the level of the intrinsic    fluorescence. This allowed to set a gate (P1) in the dot plots which    defined the level from which cells are considered expressing the    eGFP protein. Every dot inside the P1 gate was defined as an eGFP    expressing cell with a specific fluorescence intensity. The result    is shown in FIG. 12.-   b) When transfected with unmodified eGFP mRNA (A) almost all the    cells with P1 equal to 96.5% were expressing the fluorescent protein    (red population). The results are shown in FIG. 13. A very similar    result was obtained when HeLa cells were transfected with alkyne    modified eGFP mRNA (B) and a P1 value of 96.4%. The results of this    experiment are shown in FIG. 14.-   c) When HeLa cells were transfected with Eterneon Red eGFP mRNA (C)    a P1 population of 75% was observed, meaning that even by attaching    a sterically demanding dye molecule to the eGFP mRNA the ribosomes    are still able to translate it into a functional protein with 78%    relative expression level as compared to the unmodified eGFP mRNA.    Results can be seen in FIG. 15.-   d) Furthermore, because the mRNA was labelled with the Eterneon Red    dye it was possible to observe the relative mRNA amount per cell.    When the cells defined as not expressing eGFP were analysed (gate P2    in light grey) it was observed that all of them correspond to the    cells that did internalize low amounts of Eterneon Red labelled    mRNA. This assumption derives from the Eterneon Red channel where P2    (light grey) corresponds to the lowest values of fluorescence    intensity. The results are shown in FIG. 16.

1. Modified messenger RNA (mRNA), comprising a 5′-cap structure, a5′-untranslated region (5′-UTR), an open reading frame region (ORF), a3′-untranslated region (3′-UTR) and a poly(A) tail region, characterizedin that it contains at least one of an alkyne- or azide-modification inat least one nucleotide within at least one of the ORF, the 5′-UTR, the3′-UTR and the poly(A) tail region.
 2. Modified mRNA according to claim1, characterized in that it contains modified nucleotides in a) the ORFand the UTRs, b) the ORF, the UTRs and the poly(A) tail, or c) only thepoly(A) tail.
 3. Modified mRNA according to claim 1 or 2, wherein atleast one of the four standard types of nucleotides (AMP, CMP, GMP, UMP)are partly or completely modified, preferably ethynyl- or azido-modifiedat uracil or adenine.
 4. Modified mRNA according to anyone of claims 1to 3, wherein at least one nucleotide is alkyne-modified and at leastone nucleotide is azide-modified.
 5. Modified mRNA according to any oneof the preceding claims, wherein at least one of the four standard typesof nucleotides is present in modified form compared to the non-modifiedform in a ratio of 1:100 to 10:1, preferably 1:10 to 1:10 or 1:1. 6.Modified mRNA according to any one of the preceding claims,characterized in that it contains otherwise modified natural orartificial nucleotides, preferably pseudouridine orN1-methylpseudouridine.
 7. Modified mRNA according to any one of thepreceding claims, wherein the modified mRNA contains one or more of adetectable label and a functional molecule introduced via a clickreaction of the modified mRNA with a correspondingly modified alkyne- orazide-containing detectable label or functional molecule.
 8. ModifiedmRNA according to claim 7, wherein the detectable label is a colored orfluorogenic molecule and/or the functional molecule is a tissue or cellspecific targeting group or ligand, preferably a sugar moiety or a fattyacid moiety.
 9. Modified RNA containing at least one alkyne- orazide-modification in at least one nucleotide or modified mRNA accordingto anyone of claims 1 to 8, which is complexed with a cationic orpolycationic compound.
 10. Process for the production of the modifiedmRNA according to any one of the preceding claims, wherein the processcomprises in vitro transcribing mRNA from a DNA template oralternatively performing a fermentation process using prokaryotic oreukaryotic host cells to express a DNA template contained in anexpression vector wherein the process is performed in the presence of anRNA polymerase and a nucleotide mixture containing the four standardtypes of nucleotides required for mRNA transcription, in whichnucleotide mixture at least a part of at least one of the four types ofnucleotides is modified to contain an alkyne- or azide-modification. 11.Process for the production of a modified mRNA containing an alkyne- oran azide-modification at the poly(A) tail, wherein the process comprisesperforming a poly(A) polymerase addition reaction at the poly(A) tail onan mRNA in the presence of ATP, wherein ATP is at least partly alkyne-or azide-modified at the adenosine.
 12. Process according to claim 10 or11, further comprising adding a correspondingly alkyne- orazide-modified detectable label and/or functional molecule underconditions to perform a click reaction to produce a modified mRNAaccording to claim 7 or
 8. 13. Cell, which is obtained by ex vivotransfection of a corresponding human, animal or plant parent cell witha modified mRNA according to anyone of claims 1 to
 9. 14. Cell accordingto claim 13, wherein the cell is a cell of the human or animal immunesystem.
 15. Pharmaceutical composition, comprising a modified mRNAaccording to anyone of claims 1 to 9 ora cell according to claim 13 or14 as an active agent, optionally in combination with a pharmaceuticallyacceptable adjuvant or excipient and/or contained in pharmaceuticallyacceptable carrier.
 16. Modified mRNA according to anyone of claims 1 to9 or a pharmaceutical composition according to claim 15 for use in mRNAbased therapeutic and/or prophylactic applications.
 17. Modified mRNAaccording to anyone of claims 1 to 9 or pharmaceutical compositionaccording to claim 15, especially use in therapeutic and/or prophylacticapplication according to claim 16, wherein the therapeutic and/orprophylactic application comprises targeted delivery in gene replacementtherapy, targeted gene therapy in combination with specificendonucleases encoded by the mRNA (e.g. CRISPR/Cas9), in vaccination, incancer therapy and for cell specific gene expression or gene editing fortreatment of (inherited) diseases and genetic aberrations, or the use asan immunological adjuvant.
 18. Modified mRNA according to anyone ofclaims 1 to 9 or pharmaceutical composition according to claim 15,especially for use in therapeutic and/or prophylactic applicationaccording to claim 16 or 17 in a human or an animal.
 19. Use of amodified mRNA according to anyone of claims 1 to 9 for transfection ofplants and plant cells.
 20. A kit for production and/or delivery of amodified mRNA according to anyone of claims 1 to
 9. 21. Diagnosticcomposition for the in vitro screening for the presence, delivery and/ordistribution of the modified mRNA according to anyone of claims 1 to 9in cells, tissue or organs, wherein the composition contains an mRNAincluding a detectable label, preferably a fluorophore label or aradionuclide label.
 22. Diagnostic composition for use in in vivoscreening for the presence, delivery and/or distribution of the modifiedmRNA according to anyone claims 1 to 9 or for in vivo monitoring of there-administration of a cell according to claim 13 or 14, wherein thecomposition contains a modified mRNA including a fluorophore label or aradionuclide label, or the cell is transfected by a modified mRNAincluding a fluorophore label or a radionuclide label.
 23. Method forstabilizing RNA, especially mRNA, wherein an alkyne- and/or anazide-modification is introduced by including at least one of the fourstandard types of nucleotides (ATP, CTP, GTP and UTP) in partly orcompletely alkyne- and/or azide-modified form during RNA synthesisand/or in a poly(A) polymerase addition reaction to produce a modified(m)RNA, and optionally one or more of a detectable label and afunctional molecule are introduced via a click reaction of the modified(m)RNA with a correspondingly modified alkyne- or azide-containingdetectable label or functional molecule.
 24. In vitro method forqualitatively or quantitatively determining delivery and transfection ofa modified mRNA according to any one of claims 1 to 9 to target cellsvia a fluorescence-activated cell scanning analysis, which modified mRNAcontains one or more fluorogenic molecules introduced via a clickreaction to the modified mRNA with a correspondingly modified alkyne- orazide-containing fluorogenic molecule and/or which modified mRNA encodesa fluorescent protein.
 25. In vitro-method according to claim 24,wherein the fluorescence signals emitted by the fluorogenic molecule orthe fluorescent protein are determined for target cells transfected withthe modified mRNA and compared to non-transfected target cells.